Download Intrinsic cardiac ganglia and acetylcholine are important in the

Basic Res Cardiol (2017) 112:11
DOI 10.1007/s00395-017-0601-x
ORIGINAL CONTRIBUTION
Intrinsic cardiac ganglia and acetylcholine are important
in the mechanism of ischaemic preconditioning
J. M. J. Pickard1 • N. Burke1 • S. M. Davidson1 • D. M. Yellon1
Received: 24 October 2016 / Revised: 5 December 2016 / Accepted: 3 January 2017
Ó The Author(s) 2017. This article is published with open access at Springerlink.com
Abstract This study aimed to investigate the role of the
intrinsic cardiac nervous system in the mechanism of
classical myocardial ischaemic preconditioning (IPC).
Isolated perfused rat hearts were subjected to 35-min
regional ischaemia and 60-min reperfusion. IPC was
induced as three cycles of 5-min global ischaemia–reperfusion, and provided significant reduction in infarct size
(IS/AAR = 14 ± 2% vs control IS/AAR = 48 ± 3%,
p \ 0.05). Treatment with the ganglionic antagonist, hexamethonium (50 lM), blocked IPC protection (IS/
AAR = 37 ± 7%, p \ 0.05 vs IPC). Moreover, the muscarinic antagonist, atropine (100 nM), also abrogated IPCmediated protection (IS/AAR = 40 ± 3%, p \ 0.05 vs
IPC). This indicates that intrinsic cardiac ganglia remain
intact in the Langendorff preparation and are important in
the mechanism of IPC. In a second group of experiments,
coronary effluent collected following IPC, from ex vivo
perfused rat hearts, provided significant cardioprotection
when perfused through a naı̈ve isolated rat heart prior to
induction of regional ischaemia–reperfusion injury (IRI)
(IS/ARR = 19 ± 2, p \ 0.05 vs control effluent). This
protection was also abrogated by treating the naı̈ve heart
with hexamethonium, indicating the humoral trigger of IPC
induces protection via an intrinsic neuronal mechanism (IS/
AAR = 46 ± 5%, p \ 0.05 vs IPC effluent). In addition, a
large release in ACh was observed in coronary effluent was
observed following IPC (IPCeff = 0.36 ± 0.03 lM vs
Ceff = 0.04 ± 0.04 lM, n = 4, p \ 0.001). Interestingly,
however, IPC effluent was not able to significantly protect
& D. M. Yellon
[email protected]
1
The Hatter Cardiovascular Institute, University College
London, 67 Chenies Mews, London WC1E 6HX, UK
isolated cardiomyocytes from simulated ischaemia–reperfusion injury (cell death = 45 ± 6%, p = 0.09 vs control
effluent). In conclusion, IPC involves activation of the
intrinsic cardiac nervous system, leading to release of ACh
in the ventricles and induction of protection via activation
of muscarinic receptors.
Keywords Myocardial infarction Ischaemic
preconditioning Intrinsic cardiac nervous system
Abbreviations
AAR
Area-at-risk
ACh
Acetylcholine
AMI
Acute myocardial infarction
CFR
Coronary flow rate
CNS
Central nervous system
IPC
Ischaemic preconditioning
IRI
Ischaemia–reperfusion injury
IS
Infarct size
LAD
Left anterior descending
LVEDP Left ventricular end-diastolic pressure
mAChR Muscarinic acetylcholine receptor
MI
Myocardial infarction
nAChR Nicotinic acetylcholine receptor
TTC
Triphenyl tetrazolium chloride
NECA
50 -N-Ethylcarboxamidoadenosine
Introduction
Ischaemic preconditioning (IPC) is a powerful cardioprotective phenomenon, whereby brief cycles of ischaemia to
a coronary bed renders it less susceptible to subsequent
ischaemia-and-reperfusion-mediated infarction [31, 51].
Indeed, IPC has emerged as a highly conserved
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cardioprotective intervention, effective in many mammalian species via a similar mechanistic pathway (see
meta-analysis [67] and recent review [29]). Although
studies have reported protection by classical IPC in the
setting of cardiac surgery [32], it is not practical to be used
clinically in either this setting or indeed in the in the setting
of acute myocardial infarction (AMI). However, the
potency of cardioprotection offered by classical IPC provides a very useful tool to better understand the physiological basis of cardioprotection.
There are three interesting mechanistic traits of classical
IPC: (1) there are several triggers which initiate the protective reflex; (2) a threshold exists that must be surpassed
in order for protection to occur and (3) the presence of an
effector signalling pathway within the cardiomyocyte is
necessary. The triggers for IPC are several small molecules, released in the heart following the brief cycles of
ischaemia. These include adenosine [45], opioids
[14, 60, 61] and bradykinin [66]. Blocking the receptor for
one of these molecules abrogates IPC; however, this can be
overcome by additional cycles of brief ischaemia [21].
Thus, IPC involves release of multiple trigger molecules
that, via receptor activation, converge on a common must
be reached to induce cardioprotection, relating to the
strength of the IPC stimulus. Protection is observed after a
single ischaemic episode of 2.5 min, but not after shorter
periods [42]. Moreover, the strength of protection seems to
increase with the number of cycles of IPC, such that three
cycles of 5-min ischaemia affords greater protection than
one [67]. Unsurprisingly, very long IPC ischaemic cycles
no longer provide any cardioprotection [67]. Finally, the
effector pathway, which involves activation of several
well-characterised pro-survival signalling pathways within
the cardiomyocyte [27, 28] and renders the cell resistant to
death. Perhaps the most interesting aspect of IPC is that,
despite application of the intervention prior to the index
ischaemia, the majority of protection is provided against
reperfusion injury. Indeed, we have demonstrated that
inhibition of the RISK pathway at the point of reperfusion
abrogated IPC-mediated cardioprotection [26]. This
necessitates a memory phase, during which the myocardium ‘‘remembers’’ the protective intervention prior to
its employment at reperfusion. Indeed, the initial window
of protection offered by IPC lasts up to 2 h prior to the
index ischaemia [41, 44]. The mechanism of this apparent
memory phase is as yet unclear.
Neural control of the heart is typically thought to be
mediated by regions in the brainstem and spinal cord.
Indeed, the autonomic ganglia that reside within the thorax
and myocardium have long been thought of as monosynaptic relay stations, which serve to confer the complex
processing and efferent output of the central nervous system. In fact, there exists a complex hierarchy of cardiac
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Basic Res Cardiol (2017) 112:11
neural control, with sensory afferent nerves of cardiac
origin found not just in central nervous system (CNS)
ganglia, but also intrathoracic and intracardiac ganglia
[2, 22]. Intrinsic cardiac ganglia are thus able to process
sensory information and control efferent post-ganglionic
autonomic firing within the heart, in the absence of any
central modulation [1]. Moreover, a recent study revealed a
heterogeneous population of intrinsic cardiac nerves, in
particular local circuit neurons, which respond to a variety
of stimuli and can influence cardiac function on a beat-tobeat basis without CNS influence [7]. Thus, complex neural
processing occurs within the heart, not just in response to
central efferent input, but also sensory afferent information
from the myocardium. Whether these reflexes remain intact
in the Langendorff isolated heart preparation, however, is
yet to be investigated.
The massive sensory and ischaemic trauma associated
with myocardial infarction (MI) induces dynamic morphological and phenotypic remodelling of the intrinsic
cardiac nervous system, not limited to the infarcted
region [25]. A ‘neural sensory border zone’ of infarction
appears, with those afferents within the infarcted region
becoming less sensitive, and those in the border and
remote regions preserved or enhanced [57]. The influence of this neural remodelling is not yet clear, although
it is thought to contribute to ventricular arrhythmogenesis [13]. In addition, these effects occur over a period of
weeks following infarction, thus the acute influence of
the intrinsic cardiac nervous system on IRI is yet to be
understood.
The intrinsic cardiac nervous system has recently been
implicated in the cardioprotection induced by remote
ischaemic conditioning and vagus nerve stimulation
[8, 55]. Here, we present two separate studies designed to
investigate the importance of intrinsic cardiac ganglia in
classical IPC; the first using an isolated perfused heart
preparation, the second using a model of IPC via coronary
effluent transfer.
Materials and methods
Materials
Dose justification was given in detail for hexamethonium
and atropine in a recent publication from the same authors
[55]. Briefly, hexamethonium (Sigma-Aldrich, Missouri,
USA) was employed as a neuronal nicotinic acetylcholine
receptor (nAChR) antagonist, at 50 lM, to achieve specificity at nAChRs within cardiac ganglia. Atropine, a muscarinic acetylcholine receptor (mAChR) antagonist, was
used at a dose of 100 nM based on its affinity to the
receptor (Kd = 0.36 nM).
Basic Res Cardiol (2017) 112:11
Animals
All animals received humane care in accordance with the
United Kingdom (Scientific Procedures) Act of 1986. Male
Sprague–Dawley (SD) rats were bred at a central animal
unit in University College London and were used at a
weight of 250–300 g throughout the study.
Langendorff perfused heart preparation
Rats were anaesthetised with an upper left quadrant
intraperitoneal injection of sodium pentobarbitone
(60 mg/kg) (Animalcare, York, UK). Hearts were quickly
excised via a clamshell thoracotomy and the aorta cannulated on a Langendorff apparatus to allow for retrograde perfusion of modified Krebs–Henseleit buffer
(118 mM NaCl, 25 mM NaHCO3, 11 mM D-glucose,
4.7 mM KCl, 1.22 mM MgSO47H2O, 1.21 mM KH2PO4
and 1.84 mM CaCl22H2O. The buffer was warmed to
37.5 °C and gassed with 95% O2/5% CO2 to obtain a pH
of 7.35–7.45) (for detailed methods see [9]). A fluid-filled
latex balloon was inserted into the left ventricle to allow
for measurement of functional parameters, including heart
rate (HR) and left ventricular developed pressure
(LVEDP). Coronary flow rate (CFR) was recorded
throughout the protocol and the temperature of the heart
was maintained at 37.0 ± 0.5°C. Finally, a 3-0 Mersilk
suture (Ethicon, Edinburgh, UK) was inserted through the
heart to surround the left anterior descending (LAD)
coronary artery. All hearts received a 35-min LAD
ischaemia and 60-min reperfusion.
Study 1
Classical ischaemic preconditioning
Two separate experiments were designed to investigate
intrinsic cardiac nerves in classical ischaemic conditioning
(Fig. 1). The first experiment tested the involvement of
intrinsic cardiac ganglia in IPC, via use of the nicotinic
acetylcholine receptor (nAChR) antagonist, hexamethonium. Isolated perfused rat hearts were randomly assigned
to one of the following 4 groups: (1) sham IPC, hearts
received a 40-min stabilisation period; (2) control ? hexamethonium (50 lM), hearts received a 10-min stabilisation period followed by 35-min perfusion with 50 lM
hexamethonium. (3) IPC3, hearts received three cycles of
5-min global ischaemia with intermittent 5-min reperfusion
immediately prior to index ischaemia; (4) IPC3 ? hexamethonium (50lM), same as group 4, however, the hearts
were treated with hexamethonium for 5 min prior to and
the duration of the 3-cycle preconditioning.
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The second experiment examined the importance of
muscarinic acetylcholine receptors (mAChR) in classical
IPC, via use of the drug atropine. Hearts were randomly
assigned to one of 3 groups: (1) control ? atropine
(100 nM), hearts received a 10-min stabilisation followed
by 35-min perfusion with 100 nM atropine; (2) IPC3,
hearts received three cycles of 5-min global ischaemia with
intermittent 5-min reperfusion immediately prior to index
ischaemia; (3) IPC3 ? atropine (100 nM), same as group 2,
however, hearts were perfused with atropine for 5 min
prior to and the duration of the IPC protocol.
All hearts subsequently received 35-min LAD ischaemia
and 60-min reperfusion. At the end of the protocol, hearts
were analysed for infarct size using methods described
below.
Study 2
Classical IPC with coronary effluent transfer
to naı̈ve isolated hearts
This study uses a model first pioneered by the Przyklenk
laboratory [17], and has been used in several subsequent
publications by different groups [11, 43]. Although it is
described in the literature as more similar to remote
ischaemic preconditioning, in fact it likely reflects the
humoral aspect to classical preconditioning. That is, it
enables one to investigate the factors released by the heart
following IPC. RIC is now generally agreed to occur via a
more complex neuro-humoral pathway [55].
In the first part of the experiment, coronary effluent was
collected from isolated perfused donor rat hearts, randomised into one of the following two groups: (1) donor
control hearts underwent 30 min of perfusion during which
effluent was collected; (2) donor IPC hearts received three
cycles of 5-min global ischaemia with intermittent 5-min
reperfusion, during which effluent was collected.
In the second part, recipient hearts were perfused with
effluent (from above) for 10 min, following 30 min of
stabilisation, immediately prior to 35-min LAD ischaemia
and 60-min reperfusion. These recipient hearts were randomised to one of four groups: (1) Ceff, hearts received
10-min perfusion of donor control effluent immediately
prior to ischaemia; (2) IPCeff, hearts received a matched
10-min perfusion of donor IPC effluent; (3) Ceff ? Hex, the
same as group 1, however, hearts were perfused with
hexamethonium (50 lM) for 5 min prior to and the duration of effluent perfusion; (4) IPCeff ? Hex, same as group
2, however, hearts were perfused with hexamethonium for
5 min prior to and the duration of effluent perfusion. Following reperfusion, all hearts were analysed for infarct size
using methods described below.
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Basic Res Cardiol (2017) 112:11
Study 1
A. IPC + hexamethonium protocols
35’
45’
Control
60’
LAD ischemia
Stabilisation
Reperfusion
TTC staining
Hex 50uM
Control + Hex
35’
60’
5’
35’
60’
5’
35’
60’
35’
60’
35’
60’
35’
60’
45’
IPC3
15’
5’
IPC3 + Hex
15’
5’
5’
Hex 50uM
5’
B. IPC + atropine protocols
Atropine 100nM
Control + Atropine
45’
IPC3
15’
5’
IPC3 + Atropine
15’
5’
5’
5’
Atropine 100nM
5’
5’
5’
5’
Study 2
A. IPC effluent - naive isolated hearts
Control - Donor
45’
Collect coronary effluent
IPC3 - Donor
15’
5’
Perfuse coronary effluent
Recipient
35’
10’
35’
60’
35’
60’
Hex 50uM
Recipient + Hex
35’
10’
LAD ischemia
Stabilisation
Reperfusion
TTC staining
B. IPC effluent - naive isolated cardiomyocytes
Stimulate cells with effluent/drug
Normoxia
10’
Plate cells 24hr
prior to experiment
180’
240’
Normoxia
Analyse % cell death
Stimulate cells with effluent/drug
Hypox-Reox
Plate cells 24hr
prior to experiment
123
10’
Hypoxia
180’
60’
Reoxygenation
Analyse % cell death
Basic Res Cardiol (2017) 112:11
b Fig. 1 Schema detailing the experimental protocols: rat hearts were
subjected to 35-min LAD ischaemia and 60-min reperfusion.
Preconditioning was induced by three cycles of 5-min global
ischaemia–reperfusion. Study 1, hexamethonium (1a) and atropine
(1b) were perfused through the heart for 5 min prior to and for the
duration of the conditioning protocol. Study 2, coronary effluent was
collected from isolated hearts either following IPC or control, and
subsequently perfused through (2a) a naı̈ve isolated heart and prior to
index ischaemia. Again, hexamethonium was perfused through the
recipient heart for 5 min prior to and the duration of effluent
perfusion. Coronary effluent was used to stimulate isolated cardiomyocytes (2b) prior to hypoxia-reoxygenation injury
Acetylcholine assay
A Choline/Acetylcholine Assay Kit (Abcam, UK) was used
to measure the concentration of acetylcholine in effluent
collected following IPC (3 9 5-min global ischaemia–
reperfusion) or corresponding control period, as described
above. The assay was carried out in accordance with the
instructions provided by the manufacturer. Briefly, via the
use of acetylcholinesterase, the level of free and total
choline was measured in each sample, enabling an estimation of the concentration of ACh within the sample.
Classical IPC with coronary effluent transfer
to naı̈ve isolated cardiomyocytes
In order to ascertain the role of the intrinsic cardiac nerves
in classical IPC we undertook a series of studies using the
isolated cardiomyocyte, where nerves are not present.
Isolation of adult male Sprague–Dawley rat (250–300 g)
cardiomyocytes was performed using a previously described protocol [30]. Cells were plated on laminin-coated
35-mm dishes (VWR international, PA, USA) and left to
stabilise for 24 h prior to use. Dishes were assigned to one
of the following groups: (1) normoxia, cells were left in
M119 media for the duration of the protocol; (2) vector
control, cells were stimulated for 10 min with Krebs–
Henseleit buffer; (3) Ceff, cells were stimulated for 10 min
with control effluent; (4) IPCeff, cells were stimulated for
10 min with IPC effluent; (5) NECA, cells were stimulated
with the adenosine A2B receptor agonist, 50 -N-ethylcarboxamidoadenosine (NECA). Following stimulation
(groups 2–5), cells were treated with hypoxic buffer (NaCl
127.8 mM, 14.8 mM KCl, KH2PO4 1.2 mM, MgSO4
1.2 mM, NaHCO3 2.2 mM, CaCl2 1 mM, Na. lactate
10 mM, gassed with 5% CO2–95% N2 to achieve pH 6.4),
and placed into a sealed hypoxic chamber (BillupsRothenberg, CA, USA) filled with 5% CO2–95% N2 gas
mix. Hypoxia was continued for 3 h at 37 °C, at which
point the cells were removed from the chamber and treated
with normoxic buffer (glucose 10 mM, NaCl 118 mM,
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KCl 2.6 mM, KH2PO4 1.2 mM, MgSO4 1.2 mM, NaHCO3
22 mM, CaCl2 1 mM, gassed with 5% CO2–95% O2 to
achieve pH 7.4) to simulate reperfusion. The reoxygenation
was continued for 1 h, at which point the proportion of cell
death was measured via propidium iodide staining and
confocal microscopy (previously described here [65]).
Infarct size assessment
Infarct size of each isolated heart in the above experiments
(Study 1 and 2) was calculated using the following methods, described in detail previously [9]. Briefly, at the end of
the reperfusion period, the LAD suture was re-tightened
and 1 ml of 0.25% Evans blue dye was perfused through
the heart in order to delineate the area-at-risk of infarction.
The hearts were then frozen at -20°C before being sectioned into 5 transverse slices and stained for viable tissue
by immersion in 1% triphenyl-tetrazolium chloride at 37°C
for 15 min. Following fixation in 10% formalin for 24 h,
the sections were digitally scanned to a computer for
analysis. Analysis of infarct size (IS) as a proportion of
area at risk (AAR) was calculated via planimetry using
imageJ software (version 1.45, National Institutes of
Health, USA).
Statistical analysis
Data groups were first analysed for normality using the
Kolmogorov–Smirnov test. Statistical differences between
two groups were analysed using a Student’s t test and more
than two groups using a one-way analysis of variance
(ANOVA) with Tukey’s multiple comparison post-test. All
data are presented as mean ± standard error of the mean
(SEM). Data groups were classed as significantly different
with a p value less than 0.05. Notation of significance is
described in figure legends. Analyses were performed
using GraphPad Prism version 5 for Windows (CA, USA).
Results
Study 1: classical ischaemic preconditioning is
abrogated by hexamethonium and atropine
In our isolated perfused rat heart model we demonstrated
that three cycles of IPC was effective at reducing infarct
size relative to control (IS/AAR = 14 ± 2% vs control IS/
AAR = 48 ± 3%, p \ 0.05) (Fig. 2a). The nAChR
antagonist, hexamethonium (50 lM), almost fully abrogated this cardioprotection (IS/AAR = 37 ± 7%, p [ 0.05
vs control). Hexamenthonium alone did not influence
infarct size (IS/AAR = 44 ± 4%).
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*
*
80
ns
80
*
60
*
40
0
Study 2: factors released following classical IPC
require intrinsic cardiac nerves to induce protection
Effluent collected from hearts following classical IPC
induced significant protection when perfused through a
second or naı̈ve isolated rat heart prior to acute IRI (IS/
ARR = 19 ± 2,
p \ 0.05
vs
control
IS/
AAR = 46 ± 6%). Pre-treatment of the naı̈ve recipient
heart with the nicotinic antagonist, hexamethonium, abrogated the protection offered by IPC effluent (IS/
AAR = 46 ± 5%, p \ 0.05 vs IPCeff) (Fig. 3a).
A large release of ACh following IPC was observed in
these isolated perfused rat hearts, with a tenfold increase in
the concentration relative
to control effluent
(IPCeff = 0.36 ± 0.03 lM vs Ceff = 0.04 ± 0.04 lM,
n = 4, p \ 0.001) (Fig. 3b). Three of the four control
effluent samples did not contain any detectable ACh.
Classical IPC effluent appears not to protect isolated
cardiomyocytes from simulated IRI
Cells that were maintained under normoxic conditions
throughout the experiment exhibited 27 ± 2% cell death
(Fig. 4). In cells that underwent simulated IR, this was
IPC3 + Hex
0
IPC3
20
Control + Hex
20
IPC3
40
Control + Atropine
I/AAR %
60
In the second part of this study, the muscarinic antagonist, atropine, was used to investigate the pathway
downstream of intrinsic ganglia. 100 nM atropine did not
affect infarct size (IS/AAR = 51 ± 3%); however, it
abrogated the cardioprotection induced via three cycles of
IPC (IS/AAR = 40 ± 3% vs IPC = 15 ± 2%) (Fig. 2b).
123
B
*
IPC3 + Atropine
A
Control
Fig. 2 Hexamethonium and
atropine abrogate ischaemic
preconditioning:
a Hexamethonium abrogates
preconditioning induced by both
one and three cycles of IPC
(n = 6–8 per group,
asterisk = p \ 0.05 vs control);
b atropine also abrogates the
protection afforded by IPC3
(n = 6 per group except IPC ?
atropine where n = 5,
asterisk = p \ 0.05 vs control
? atropine). Data presented as
mean ± SEM
Basic Res Cardiol (2017) 112:11
I/AAR %
11
increased to 57 ± 6 and 64 ± 6% after pre-treatment with
the vehicle control and control effluent, respectively
(p \ 0.001 vs normoxic in both cases). Treating the cells
with IPC effluent did not significantly reduce cell death
(45 ± 6%, p = 0.09 vs Ceff). The adenosine A2b agonist
NECA (used as a ?ve control) significantly reduced cell
death to 32 ± 4% (p \ 0.01 vs vector control and Ceff)
(Fig. 4).
Discussion
These results are the first indication of a neural pathway in
the mechanism of classical ischaemic preconditioning. We
demonstrated an important role for nicotinic and acetylcholine receptors, suggesting that intrinsic cardiac ganglia
remain active in the isolated heart preparation and are
important in conveying the protective message. Acetylcholine is released from the heart following IPC, perhaps
from parasympathetic post-ganglionic nerve endings in the
ventricles, and induces protection via an atropine-sensitive
mechanism. The lack of total abolition of protection, in the
presence of ganglionic or muscarinic antagonism, is likely
due to the fact that several factors contribute to the IPC
mechanism in the isolated heart model [18]. Moreover,
coronary effluent collected following IPC was able to
protect a naı̈ve isolated heart, but not isolated cardiomyocytes, from IRI. The protection in naı̈ve hearts was
abrogated by hexamethonium, highlighting the importance
of intrinsic cardiac ganglia in the mechanism of IPC. We
therefore propose that IPC is governed, in part, via a neuro-
Basic Res Cardiol (2017) 112:11
Page 7 of 12
A
80
*
*
B
0.4
ACh (uM)
60
40
0.3
0.2
0.1
0
0.0
Control (eff)
IPC(eff)+hex
IPC (eff)
C(eff)+hex
20
Control (eff)
I/AAR(%)
*
0.5
IPC (eff)
Fig. 3 IPC effluent protects the
naı̈ve isolated heart from
ischaemia–reperfusion injury.
a Effluent collected following
IPC significantly protected a
naı̈ve isolated rat heart via a
hexamethonium-sensitive
mechanism (n = 7–8 per group,
asterisk = p \ 0.05 vs control
effluent); c the concentration of
ACh in coronary effluent
increases tenfold following IPC
(n = 4 per group,
asterisk = p \ 0.05 vs Control
effluent). Data presented as
mean ± SEM
11
[ACh]
Exposed to hypoxia-reoxygenation
A
ns
% Cell Death
100
*
B
*
*
Exposed to hypoxia-reoxygenation
Normoxia
VC
NECA
Ceff
IPCeff
80
60
40
NECA
IPCeff
Ceff
Vehicle Control
0
Normoxia
20
Fig. 4 IPC effluent does not protect isolated cardiomyocytes from
simulated ischaemia–reperfusion injury. a Isolated rat cardiomyocytes were not protected from hypoxia-reoxygenation injury by prior
exposure to IPC effluent. The adenosine A2B agonist, NECA, was
able to reduce cell death significantly (n = 6 in all groups except
n = 4 for NECA, asterisk = p \ 0.05 vs normoxia); b representative
images of isolated cardiomyocytes subjected to the different protocols, the red staining indicate dead cells. Data presented as
mean ± SEM
humoral pathway; a factor released following IPC activates
intrinsic cardiac ganglia, leading to release of ACh from
parasympathetic post-ganglionic nerve endings in the
ventricles, thus inducing cardioprotection via activation of
muscarinic receptors. These data are, to some extent,
additive to our previous study, where we proved an
important role for intrinsic cardiac ganglia in remote
ischaemic conditioning. Whilst a neural pathway has been
well validated in RIC, this is the first to imply a similar
intrinsic cardiac neural pathway in IPC. Thus, there appear
to be more similarities between the mechanisms of
classical (direct) and remote ischaemic conditioning than
were previously apparent [5, 55].
Intrinsic cardiac ganglia in the isolated heart
Intrinsic cardiac ganglia are widely distributed in the
myocardium and not only relay central efferent pre-ganglionic information, but also are able to process sensory
afferent information from the myocardium and control
efferent post-ganglionic firing [7, 46]. Moreover, several
anatomical and functional studies have indicated a
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significant presence of vagal neurons in the ventricles, in
addition to sympathetic and local circuit neurons [3]. The
intrinsic cardiac nervous system is therefore able to control
cardiac indices on a beat-to-beat basis, in the absence of
input from the central nervous system [4].
Activation of sensory afferent nerves in the Langendorff
heart has previously been demonstrated to induce cardioprotection. Perfusion with capsaicin, a known activator of
C-fibre afferents, induced early and delayed protection
against ischaemia–reperfusion injury in isolated Langendorff rat hearts [69]. In our study, ganglionic antagonism
abrogated the protection afforded by IPC, suggesting sensory afferent activation occurs following IPC and is
important in conferring the cardioprotection. We further
demonstrated a tenfold increase in the ACh concentration
in perfusate collected following IPC. This suggests activation of post-ganglionic parasympathetic neurones from
the intrinsic ganglia. Indeed, muscarinic antagonism, using
atropine, abolished IPC-induced cardioprotection. The
parasympathetic nervous system has a well-defined cardioprotective effect [34, 48, 49, 62], and recently emerged
as a key mediator of the cardioprotection afforded by
‘‘remote’’ ischaemic conditioning (RIC) [6, 49, 55]. Indeed,
a recent study demonstrated that increased parasympathetic
tone ameliorated the functional and structural remodelling
of the intrinsic cardiac nervous system following myocardial infarction [8]. Thus, we propose that there exist similarities between the mechanism of remote and classical
ischaemic conditioning [37, 54]; an intrinsic neural reflex
loop in response to the brief ischaemia of IPC, which
activates cardiac ganglia and increases post-ganglionic
vagal tone, leading to release of ACh in the ventricles.
A contentious role for acetylcholine in IPC
IPC is triggered via release of several small molecules, and
their subsequent receptor activation in the myocardium.
This was first demonstrated by Liu et al., who showed that
pre-treatment with an adenosine receptor antagonist abrogated IPC in rabbit hearts [45]. Moreover, perfusion of
exogenous adenosine through the heart prior to infarction
mimicked the cardioprotection of IPC. These data suggested endogenous release of adenosine occurs in response
to IPC, which protects the myocardium. Two other small
molecules, opioids [14, 47, 60] and bradykinin [66], were
found in subsequent studies to be important in the mechanism of IPC, via activation of their receptor. These appear
to be connected via a common intracellular cytoprotective
signalling pathway [53], which centres on activation of
protein kinase C (PKC) [18]. Coronary effluent collected
following IPC can protect a naı̈ve isolated heart from
infarction [11, 16], supporting the theory of a humoral
trigger for IPC. Indeed, adenosine is released into the
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Basic Res Cardiol (2017) 112:11
effluent following IPC, and confers protection to the naı̈ve
heart via crosstalk with opioid receptors [15, 43]. Bradykinin is not involved in this setting due to the requirement
for circulating kininogens in the blood, not present in the
isolated buffer-perfused model [21].
The role for acetylcholine in IPC, however, is more
contentious. While several studies from Krieg et al.
demonstrated exogenous ACh could induce cardioprotection in the Langendorff model, the same group discounts its
involvement in the mechanism of IPC [38–40]. However,
two studies from Kawada et al. demonstrated brief, 5-min
ischaemia in in vivo rabbit and cat models induces interstitial release of ACh in the ventricles [35, 36]. Data from
our study confirm those of Kawada et al. with a significant
release of ACh observed in coronary effluent following
IPC. Given this observation, we hypothesised that isolated
cardiomyocytes would be protected from simulated IRI
following exposure to coronary effluent. Presumably, if
ACh was mediating protection here, it would act directly
on muscarinic receptors on the cardiomyocytes in the naı̈ve
heart. However, IPC effluent was not able to significantly
reduce cell death in isolated cardiomyocytes subjected to
hypoxia-reoxygenation. A small reduction in cell death is
observed, likely due to the presence of several trigger
factors in the effluent [15, 43]; however, this was not statistically significant. This is perhaps due to dilution of
factors released from the myocardium in the effluent following IPC, such that the concentration in the isolated cells
would be insufficient for cardioprotection. However, we
did not investigate whether exogenous ACh of the same
concentration could induce cardioprotection in isolated
cardiomyocytes or Langendorff models.
Finally, coronary effluent collected following IPC
induced powerful cardioprotection when perfused through
naı̈ve isolated rat hearts, via a mechanism also sensitive to
hexamethonium. This experiment confirms the key point to
the study; factors released following IPC require intrinsic
cardiac ganglia to induce cardioprotection. The neural and
humoral components to IPC, therefore, are co-dependent.
Is there a common trigger for classical and remote
ischaemic conditioning?
Our data suggest that both classical and remote ischaemic
conditioning (RIC) may share a common trigger pathway;
i.e. local release of an autocoid, activation of sensory
afferent nerves and subsequent recruitment of the intrinsic
cardiac nervous system. RIC is induced via the same
principle as classical IPC, brief cycles of ischaemia, however applied to a region remote from the heart [10]. The
trigger for RIC is thought to be local release of an autocoid,
such as adenosine, which activates sensory afferent nerves
communicating the protective message away from the
Basic Res Cardiol (2017) 112:11
conditioned limb. Indeed, a small injection of adenosine
into the femoral artery is sufficient to induce cardioprotection [64], as is activation of C-fibre sensory afferents by
capsaicin [58] or transcutaneous electrical nerve stimulation [50]. Moreover, our study has demonstrated the
importance of intrinsic cardiac ganglia in IPC, which
necessitates prior sensory nerve activation in the heart.
Classical IPC is known to involve release of adenosine and
calcitonin gene-related peptide [43, 45], both of which are
know to activate sensory afferent nerves. Perhaps, therefore, these are the trigger for this aspect to IPC. The trigger
for both RIC and classical IPC appear to share important
similarities, with both neural and humoral components
[55].
A recent meta-analysis revealed that ischaemic preconditioning had variable efficacy in mammalian species;
namely, IPC was more effective in rodents relative to nonrodents [67]. Myocardial autonomic innervation is known
differ according to the species’ size [59]; however, it is not
clear whether these differences relate to the speciesspecific effect of IPC. Finally, with respect to remote
ischaemic conditioning, although there appear to be differences in the signalling cascades important for cardioprotection between species [63], a recent meta-analysis
revealed no difference in the efficacy of cardioprotection
relative to species [12].
Finally, it is important to note the range of different
factors that contribute to the trigger phase of IPC, in
addition to the release of autacoids and neuro-humoral
factors. Physical stimuli, such as activation of stretch
receptors during the preconditioning stimulus, have been
demonstrated to reduce infarct size [24]. In addition,
reactive hyperaemia during brief reperfusion induces
release of nitric oxide from endothelial cells, which can
trigger IPC, although this effect appears to be species
specific [52, 56]. Several other intracellular chemical
stimuli have been implicated in the trigger phase of IPC.
The brief ischaemia and reperfusion of IPC induces release
of reactive oxygen species in cardiomyocytes, which in
small quantities can induce cardioprotection [31]. Thus,
IPC is triggered via a variety of stimuli, all of which
contribute to reaching the appropriate threshold for cardioprotection to ensue.
Study limitations
The key limitation of the study lies in the specificity of
hexamethonium to neuronal nicotinic acetylcholine receptors. Hexamethonium is widely used within the literature as
a ganglionic blocker, being a neuronal nicotinic acetylcholine receptor antagonist. Although it has some affinity
to muscarinic M2 receptors, this becomes negligible at
concentrations below 100 lM [20], and it has an IC50 of
Page 9 of 12
11
11 lM at nAChRs [23]. Thus, for the purposes of this study
50 lM was used in order to achieve high specificity to
nicotinic receptors, but the potential may remain for nonspecific effects. The second issue relates to the spatial
expression of nicotinic receptors within the myocardium.
Although nicotinic receptors are largely limited to the
intrinsic cardiac ganglia, there is evidence for expression of
the receptors on the myocytes [19]. Thus, there is a small
possibility that hexamethonium is exerting its effect
through non-ganglionic action.
Secondly, these data do not fully ascertain the role of
ACh within the effluent. An important additive experiment
would be to treat a naı̈ve isolated heart with acetylcholine
at the same level observed within the effluent. In addition,
it is possible that stimulation of isolated cardiomyocytes
with effluent for longer than 10 min could reveal
cardioprotection.
Conclusion
This is the first study to implicate intrinsic cardiac ganglia
in the mechanism of classical ischaemic conditioning. We
propose that IPC activates an intrinsic cardiac neural reflex
and is an important part of the cardioprotective mechanism.
This is a significant finding for several reasons. The Langendorff perfused heart was traditionally thought of as a
denervated preparation; however, clearly this is not true
given the current data, in addition to our recent publication
[55]. Moreover, these data add to the paradigm that IPC is a
receptor-mediated phenomenon. However, there seems to
be an added layer of complexity, with the intrinsic ganglia
responsible for conferring a portion of the cardioprotection.
This is of importance given the issue of co-morbidities in
the clinical setting. For example, diabetes has been well
documented to decrease the efficacy of IPC [68]; perhaps
this could be explained by the peripheral sensory neuropathy that occurs as the disease progresses [33]. Whether
the peripheral sensory nerve activation induced via remote
ischemic conditioning is comparable to that of classical
ischaemic conditioning is not clear. Further work is necessary to ascertain the exact nature of the involvement of
intrinsic ganglia in this setting.
Acknowledgements The authors thank Prof. Alex Gourine (UCL)
for his help in the initial intellectual discussions regarding intrinsic
cardiac ganglia and ischaemic preconditioning.
Open Access This article is distributed under the terms of the
Creative Commons Attribution 4.0 International License (http://crea
tivecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided you give
appropriate credit to the original author(s) and the source, provide a
link to the Creative Commons license, and indicate if changes were
made.
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